The role of blood coagulation is to provide an insoluble fibrin matrix for consolidation and stabilization of a haemostatic plug. Formation of a cross-linked fibrin clot results from a series of biochemical interactions involving a range of well-characterized plasma proteins.
The interactions are divided into what are termed the "intrinsic pathway", in which all the substances necessary for fibrin formation are present in precursor form in circulating plasma, and the extrinsic pathway in which thromboplastin, derived from tissues, bypasses several steps in the process and accelerates clot formation. The two pathways are highly interdependent and Factor VII, Factor IX and Factor X are mutually activated (J. C. Giddings "Molecular Genetics and Immunoanalysis in Blood Coagulation" Ellis Horwood Ltd., Chichester, England 1988, p. 17).
The role of Factor X in the coagulation cascade has been reviewed by Zur et al. "Tissue factor pathways of blood coagulation", Haemostasis and Thrombosis, 1.sup.st Edition (Bloom et al., Eds) Churchill Livingstone, Edinburgh, pp. 124-139 (1981); Jackson, "The biochemistry of prothrombin activation" Haemostasis and Thrombosis, 2.sup.nd edition (Bloom et al; Eds) Churchill Livingstone, Edinburgh, pp. 165-191 (1987) and Steinberg et al. "The activation of Factor X" Haemostasis and Thrombosis, First Edition (Colman et al., Eds.) Lippincott, Philadelphia, pp. 91-111 (1982).
Human Factor X circulates in plasma as a two-chain glycoprotein with a molecular weight of about 67000 estimated by gel electrophoresis in the presence of sodium dodecylsulphate (Di Scipio et al., "A comparison of human prothrombin, Factor IX (Christmas factor), Factor X (Stuart factor) and protein S", Biochemistry, vol. 16, pp. 698-706). Slightly lower Mr values of about 59000 are obtained by sedimentation equilibrium analysis. The normal plasma concentration is about 7-10 mg per liter and the protein contains about 15% carbohydrate.
The heavy chain of Factor X, Mr=42000 demonstrates a high degree of homology with prethrombin 2 and contains the active site serine. It is covalently linked to the light chain, Mr=17000, by disulphide bridges. The light chain contains the alpha-carboxylated glutamic acid residues and shows significant homology with prothrombin fragment 1. Activation of Factor X by complexes of Factor IXa and Factor VIIIa or by tissue factor/Factor VIIa involves at least two peptide cleavages. The principal mechanism releases a small activation peptide from the heavy chain of the molecule by hydrolysis of an arginine-isoleucine bond. The active product formed in this way is termed alpha-factor Xa. This is modified further by cleavage of an arginine-glycine bond near the carboxy terminus to form .beta.-factor Xa. Both alpha-Xa and .beta.-Xa have the same coagulation activity.
The light chain of Factor X is unaffected by the activation process and remains linked to the heavy chain of the molecule by disulphide bridges. In this way the active substance retains the gamma-carboxyglutamic acid domains necessary for calcium-mediated attachment to phospholipid micelles or cellular surfaces. Fundamentally, therefore, in contrast to thrombin, Factor Xa remains associated with phospholipid and platelet membranes.
U.S. Pat. No. 4,588,587 describes the anticoagulant activity of Haementeria officinalis leech saliva.
Vermulen et al., Int. J. Biochem, Vol. 20, No. 6, pp. 621-31 (1988), describes the relative protease inhibition activities of tick toxins isolated from R. evertsi evertsi, B. decoloratus, B. microplus, and H. truncatum. They were found to be fast-binding or slow-binding inhibitors of trypsin and chymotrypsin.
Willadsen and Riding, Biochem J., Vol. 189, pp. 295-303 (1980), describe activity of a proteolytic-enzyme inhibitor from the ectoparasitic tick Boophilus microplus, and the effect of the inhibitor on blood-coagulation parameters.
Prior studies identified and partially purified inhibitors of Factor Xa Markwardt, F. et al. (1958) Naturwissenschaften 45, pp, 398-399 and Markwardt, F. et al. (1961) Naturwissenschaften 48 p. 433 and thrombin Hawkins, R. et al. (1967) Proceedings of the Royal Society 70 p. in tick saliva. It has been suggested that saliva contains an inhibitor of Factor IXa (Hawkins). (Hellmann, K. and Hawkins (1967) Thromb. Diath. Haemorrh. 18 pp. 617-625) Ribeiro et al. reported that tick saliva blocks clotting by inhibiting the intrinsic pathway (Ribeiro, J. (1985) J. Exp. Med. 161 pp. 332-344) but they did not identify the site of inhibition. This study also demonstrated for the first time antiplatelet activity which blocked platelet aggregation induced by ADP, collagen, or platelet activating factor (Ribeiro, J. (1985) J. Exp. Med. 161 pp. 332-344). No attempt was made to purify any of these factors to enable an analysis of their structures or mechanism of action.
The S. cerevisiae .alpha.-mating factor pre-pro leader sequence has been utilized in the expression of heterologous genes as secreted products in yeast (Brake et al., Proc. Natl. Acad. Sci. USA vol. 81, pp. 4642-4646 (1984); Miyajima et al., Gene vol. 37, pp. 155-161 (1985); Vlasuk et al., J. Biol. Chem. vol. 261, pp. 4789-4796 (1986); Schultz et al., Gene vol. 54, pp. 113-123 (1987); Schultz et al., Gene vol. 61, pp. 123-133 (1987); Bayne et al., Gene, vol. 66 pp. 235-244 (1988); and Laison et al., Biotechnology, vol. 6, pp. 72-77 (1988)). Proteins produced as fusion products are proteolytically processed by the Lys-Arg-cleaving endopeptidase (KEX2) encoded by the KEX2 gene and products are secreted into the culture medium. The KEX2 cleaves on the C-terminal side of Lys-Arg residues which are present between the ppL sequence and the heterologous gene.